Activation of p34 super(cdc2) protein kinase by microinjection of human cdc25C into mammalian cells: Requirement for prior phosphorylation of cdc25C by p34 super(cdc2) on sites phosphorylated at mitosis

• Published in: The Journal of biological chemistry 1994-01, Vol.269 (8), p.5989-6000
• Main Authors: Strausfeld, U, Fernandez, A, Capony, J-P, Girard, F, Lautredou, N, Derancourt, J, Labbe, J-C, Lamb, NJC
• Format: Article
• Language: English
• ISSN: 0021-9258

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34 super(cdc2) protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G sub(2)-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34 super(cdc2)-cyclin B protein kinase. Of seven putative phosphorylation sites for p34 super(cdc2) protein kinase present in human cdc25C, five are phosphorylated by p32 super(cdc2) protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G sub(2)-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34 super(cdc2) protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G sub(2) cells into premature prophase in a manner similar to microinjection of purified active p34 super(cdc2) protein kinase. The data show that multisite phosphorylation of cdc25C by p34 super(cdc2)-cyclin B protein kinase occurs at the G sub(2)-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.